The invention concerns the production of heterologous proteins in yeast.
Yeast organisms are favored for producing heterologous proteins by recombinant DNA technology due to their ability to secrete expressed proteins from the cell. Secretion of the produced protein has several advantages. First, secretion avoids cellular toxicity, which is often a problem when recombinant proteins accumulate inside the cell. Second, many proteins require passage through the secretory pathway in order to obtain proper conformational folding and full biological activity. Third, biochemical purification of the recombinant protein is facilitated by secretion since higher initial degrees of purity are obtained in the culture broth than in a cell lysate, and fewer purification steps are needed.
Transport of proteins through the cellular organelles of the secretory pathway is dependent in part on having an N-terminal secretion sequence. In yeast this sequence takes the form of a signal peptide which is synthesized as part of the precursor form of the protein. The signal peptide, removed by specific proteolytic cleavage during transit, is not part of the mature protein product. The signal peptide is found at the extreme N-terminus of the precursor form of the protein and mediates translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum (Walter et al. 1984 Cell 38:5-8; Nicchitta et al. 1991 Cell 65:587-598; Benson et al. 1985 Ann. Rev. Biochem. 54:101-134). The signal peptide is often referred to in the literature as a "pre-peptide." A signal peptidase cleaves off the signal peptide during translocation within the endoplasmic reticulum. In the yeast Saccharomyces cerevisiae, the signal peptidase is the product of the SEC11 gene (Bohni, et al 1988 J. Cell Biol. 106:1035-1042).
For many secreted proteins, cleavage of the signal peptide results in production of the mature form of the protein. However, there are other secreted proteins, including, e.g., human insulin-like growth factor-I (IGF-I), that require, when they are to be secreted by yeast cells, a sequence between the carboxy-terminus of the signal peptide and the amino-terminus of the mature protein that is commonly known as a "pro" sequence. In the absence of a pro sequence some proteins are retained as intracellular, inactive precursors in the cell (Chaudhuri et al. 1992. Eur. J. Biochem. 206:793-800). The pro-protein (i.e., a protein precursor with an N-terminal pro sequence) remains intact during transport to the Golgi apparatus, but the pro sequence is subsequently removed in the trans-Golgi or secretory vesicles (Julius et al. 1984. Cell 36:309-318; Redding et al. 1991. J. Cell Biol. 113:527-538; Franzusoff et al. 1991. J. Cell Biol. 112:27-37). In S. cerevisiae, the pro sequence is removed by an endoproteinase (a product of the KEX2 gene) that is a trypsin-like protease and cleaves on the carboxyl side of a pair of dibasic amino acid residues (Julius et al. 1984. Cell 37:1075-1089).
In addition, several secreted proteins in yeast contain short spacer amino acid sequences preceding the N-terminus of the mature protein. For example, the lys-arg recognition sequence for the KEX2 protease also comprises part of a spacer sequence separating four repeats of the mature alpha-MF peptide in S. cerevisiae, and is cleaved initially by the KEX2 protease at the Arg-Glu peptide bond (Brake, 1990. Meth. Enz. 185:408-421). The remaining Glu/Asp-Ala repeats are removed subsequently by a membrane-bound dipeptidylaminopeptidase (Julius et al. 1983. Cell 32:839-852). The role the spacer sequences have in the secretion of endogenous or recombinant proteins is unclear.